Archives

  • 2026-02
  • 2026-01
  • 2025-12
  • 2025-11
  • 2025-10
  • 2023-07
  • 2023-06
  • 2023-05
  • 2023-04
  • 2023-03
  • 2023-02
  • 2023-01
  • 2022-12
  • 2022-11
  • 2022-10
  • 2022-09
  • 2022-08
  • 2022-07
  • 2022-06
  • 2022-05
  • 2022-04
  • 2022-03
  • 2022-02
  • 2022-01
  • 2021-12
  • 2021-11
  • 2021-10
  • 2021-09
  • 2021-08
  • 2021-07
  • 2021-06
  • 2021-05
  • 2021-04
  • 2021-03
  • 2021-02
  • 2021-01
  • 2020-12
  • 2020-11
  • 2020-10
  • 2020-09
  • 2020-08
  • 2020-07
  • 2020-06
  • 2020-05
  • 2020-04
  • 2020-03
  • 2020-02
  • 2020-01
  • 2019-12
  • 2019-11
  • 2019-10
  • 2019-09
  • 2019-08
  • 2019-07
  • 2019-06
  • 2019-05
  • 2019-04
  • 2018-11
  • 2018-10
  • 2018-07

    • Cy5 Maleimide (Non-sulfonated): Precision Thiol Labeling ...

    2025-11-20

    Cy5 Maleimide (Non-sulfonated): Precision Thiol Labeling for Site-Specific Protein Imaging

    Executive Summary: Cy5 maleimide (non-sulfonated) is a mono-reactive, thiol-specific fluorescent dye for covalent labeling of cysteine residues in peptides and proteins (APExBIO). It features excitation/emission maxima at 646/662 nm, a high extinction coefficient (250,000 M-1cm-1), and quantum yield of 0.2, ensuring strong, quantifiable signals for advanced fluorescence imaging (internal). The maleimide functionality guarantees selective, covalent conjugation to thiol groups under mild conditions, enabling site-specific protein modification (Chen et al., 2023). Due to low aqueous solubility, Cy5 maleimide must be pre-dissolved in DMSO or ethanol before use. The reagent is widely adopted in molecular imaging, immunotherapy research, and nanotechnology for tracking and visualizing biomolecules.

    Biological Rationale

    Cysteine residues are rare and often strategically positioned within proteins, making them valuable targets for site-specific labeling and functionalization. The thiol (-SH) group on cysteine is nucleophilic, enabling selective conjugation through maleimide chemistry under physiological pH (6.5–7.5) (internal). Covalent labeling with Cy5 maleimide (non-sulfonated) allows researchers to visualize, quantify, and track proteins and peptides in complex biological systems. This is essential for studies in protein dynamics, protein-protein interactions, and the development of targeted nanomotors or drug delivery systems (Chen et al., 2023). The use of a far-red cyanine dye minimizes background autofluorescence and photobleaching, increasing sensitivity for deep-tissue and live-cell imaging. Compared to amine-reactive NHS esters, maleimide-functionalized dyes provide superior selectivity for thiol groups, reducing off-target labeling in complex samples (internal).

    Mechanism of Action of Cy5 maleimide (non-sulfonated)

    Cy5 maleimide (non-sulfonated) contains a maleimide moiety that undergoes a Michael addition with sulfhydryl groups (-SH) of cysteine residues. The reaction proceeds efficiently at pH 6.5–7.5 and room temperature, forming a stable thioether bond. The covalent nature of this linkage ensures irreversible tagging of the protein or peptide, preserving spatial and temporal information during downstream detection (internal). The cyanine dye scaffold absorbs maximally at 646 nm and emits at 662 nm, compatible with most red/far-red fluorescence imaging platforms. Because the dye is non-sulfonated, it has low aqueous solubility, necessitating dissolution in DMSO or ethanol prior to conjugation (APExBIO). This workflow minimizes hydrolysis of the maleimide group and maximizes labeling efficiency. The resulting conjugate is stable to light and temperature if stored at -20°C in the dark.

    Evidence & Benchmarks

    • Cy5 maleimide (non-sulfonated) enables site-specific, covalent labeling of cysteine residues in proteins and peptides under physiological conditions (pH 6.5–7.5, 20–25°C) (Chen et al., 2023).
    • The dye exhibits excitation and emission maxima at 646 nm and 662 nm, respectively, minimizing background autofluorescence in biological samples (APExBIO).
    • A high extinction coefficient (250,000 M-1cm-1) and quantum yield (0.2) ensure robust signal for fluorescence microscopy and flow cytometry (internal).
    • Cy5 maleimide labeling is compatible with a wide range of imaging instruments, including confocal microscopes, plate readers, and in vivo imaging systems (internal).
    • The dye is stable for up to 24 months at -20°C in the dark, withstanding room temperature shipping for up to 3 weeks (APExBIO).
    • Reactive oxygen species concentrations in tumor microenvironments (as high as 100 μM) can be visualized using labeled nanomotors, enhancing immunotherapy research (Chen et al., 2023).

    Applications, Limits & Misconceptions

    Cy5 maleimide (non-sulfonated) is used extensively in:

    • Protein and peptide labeling for fluorescence microscopy, flow cytometry, and in vivo imaging.
    • Development of chemotactic nanomotors for targeted delivery and tracking in cancer immunotherapy (Chen et al., 2023).
    • Generation of fluorescent probes for studying protein-protein interactions and subcellular localization (internal).

    This article extends previous reviews (Cy5 Maleimide: Next-Gen Fluorescent Probe) by providing detailed quantitative parameters and clarifying optimal workflow integration for site-specific protein modification.

    Common Pitfalls or Misconceptions

    • Non-selective labeling: Cy5 maleimide only reacts with thiol groups; it does not label amines, hydroxyls, or carboxylates under standard conditions.
    • Hydrolysis risk: The maleimide group hydrolyzes rapidly in aqueous solution above pH 7.5, reducing labeling efficiency.
    • Aqueous solubility: The non-sulfonated dye has poor solubility in water and must be pre-dissolved in DMSO or ethanol.
    • Photobleaching: Prolonged exposure to light can degrade the fluorophore; store and handle in the dark.
    • Not for diagnostic use: Cy5 maleimide is strictly for research; it is not approved for clinical or diagnostic applications.

    Workflow Integration & Parameters

    For optimal labeling, dissolve Cy5 maleimide (non-sulfonated) in dry DMSO or ethanol at concentrations between 1–10 mM. Add the dye solution dropwise to a buffered protein sample (e.g., phosphate-buffered saline, pH 7.0), maintaining a final dye:protein molar ratio of 1.2:1 to 3:1. Incubate at room temperature (20–25°C) for 30–60 minutes, shielded from light. After reaction, remove excess dye by gel filtration or dialysis. Store labeled conjugates at 4°C (short term) or -20°C (long term) in the dark (APExBIO). This protocol ensures high labeling efficiency and minimal non-specific background. For advanced nanomotor engineering and immunotherapy research, site-specific labeling facilitates precise tracking in complex biological environments (internal), an update over prior generalized workflows.

    Conclusion & Outlook

    Cy5 maleimide (non-sulfonated) from APExBIO provides robust, reproducible, and site-specific labeling of thiol-containing biomolecules. Its high extinction coefficient, optimized emission/excitation, and selective maleimide chemistry make it the dye of choice for demanding imaging and protein conjugation workflows. As advanced nanotechnologies and immunotherapeutic applications expand, the ability to precisely track and quantify protein modifications using dyes like Cy5 maleimide will remain essential (Chen et al., 2023). Researchers must ensure proper handling and workflow integration to maximize performance and minimize common errors. For more details and current protocols, consult the official product documentation for Cy5 maleimide (non-sulfonated), A8139.